Restriction Enzyme Cloning

Restriction enzyme (endonuclease) based molecular cloning is the “classic” cloning method, and for many reasons, remains one of the most popular today. Restriction enzyme are used to cut double stranded DNA into fragments containing precise 5′ or 3′ single-strand overhangs (sticky ends). Two pieces of DNA that have complementary overhangs can then be fused together during a ligation reaction. Restriction enzyme cloning benefits from the hundreds of available enzymes, each with a specific target sequence. This allows synthetic biologists to design constructs with multiple unique restriction sites that can be used to replace any piece of DNA region between any two restriction sites or easily move a piece of DNA into any other plasmid harboring the same two restriction sites. The downside of restriction enzyme cloning is that you can hardly assemble more than 3 fragments simultaneously, which requires multiple cloning steps to built-up an entire plasmid.